scholarly journals Monitoring Protein Kinase Activity in Cell Lysates Using a High-Density Peptide Microarray

2009 ◽  
Vol 14 (3) ◽  
pp. 256-262 ◽  
Author(s):  
Xiaoming Han ◽  
Go Yamanouchi ◽  
Takeshi Mori ◽  
Jeong-Hun Kang ◽  
Takuro Niidome ◽  
...  
Keyword(s):  
Protein Kinase ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Disease Diagnosis ◽  
High Density ◽  
Protein Kinase Activity ◽  
Peptide Array ◽  
Screening Assay ◽  
Chip Technology ◽  
High Throughput Screening Assay

Monitoring and targeting protein kinases is widely accepted as a promising approach for disease diagnosis and drug discovery. For this purpose, the authors have developed an original type of peptide array as a high-throughput screening assay for quantitatively evaluating kinase activity. A volume of 2 nL of peptide solution was spotted onto a formyl group-modified glass slide by using an arrayer, which was designed for use with protein chip technology. The phosphorylation was recognized by fluorescence-label antibody and detected with an automatic microarray scanner widely used in DNA chip technology. The system needs low sample volume, provides a high-density peptide array, and supplies high reproducibility. It provided enough sensitivity for inhibitor screening, even though a relatively low concentration of purified kinase was employed. The assay also proved useful for the detection of intracellular kinase activity as well as for the measurement of the fluctuations of intracellular protein kinase activity with drug stimulation. Thus, this peptide array would be applicable for kinase-targeted diagnosis, cell-based drug screening, and signal pathway investigation. ( Journal of Biomolecular Screening 2009:256-262)

scholarly journals CDK10 in Gastrointestinal Cancers: Dual Roles as a Tumor Suppressor and Oncogene

2021 ◽  
Vol 11 ◽  
Author(s):  
Zainab A. Bazzi ◽  
Isabella T. Tai
Keyword(s):  
Cell Proliferation ◽  
Tumor Suppressor ◽  
Small Molecule ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Screening Assay ◽  
Cellular Processes ◽  
Molecule Inhibitor ◽  
High Throughput Screening Assay ◽  
Kinase Activity Assay

Cyclin-dependent kinase 10 (CDK10) is a CDC2-related serine/threonine kinase involved in cellular processes including cell proliferation, transcription regulation and cell cycle regulation. CDK10 has been identified as both a candidate tumor suppressor in hepatocellular carcinoma, biliary tract cancers and gastric cancer, and a candidate oncogene in colorectal cancer (CRC). CDK10 has been shown to be specifically involved in modulating cancer cell proliferation, motility and chemosensitivity. Specifically, in CRC, it may represent a viable biomarker and target for chemoresistance. The development of therapeutics targeting CDK10 has been hindered by lack a specific small molecule inhibitor for CDK10 kinase activity, due to a lack of a high throughput screening assay. Recently, a novel CDK10 kinase activity assay has been developed, which will aid in the development of small molecule inhibitors targeting CDK10 activity. Discovery of a small molecular inhibitor for CDK10 would facilitate further exploration of its biological functions and affirm its candidacy as a therapeutic target, specifically for CRC.

A robust and quantitative assay platform for multiplexed, high throughput screening of protein kinase inhibitors

2016 ◽  
Vol 52 (81) ◽  
pp. 12112-12115 ◽  
Author(s):  
Jieon Lee ◽  
Il-Soo Park ◽  
Ginam Park ◽  
Kyukwang Cho ◽  
Hee-Sung Park ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Protein Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Protein Kinase Activity ◽  
Activity Assay ◽  
Kinase Activity Assay

We present a new platform for multiplexed protein kinase activity assay using TiO2decorated graphene oxide (GO), which is applicable to high throughput inhibitor screening.

scholarly journals Microarrayed Compound Screening (μARCS) to Identify Activators and Inhibitors of AMP-Activated Protein Kinase

2004 ◽  
Vol 9 (2) ◽  
pp. 112-121 ◽  
Author(s):  
Steven N. Anderson ◽  
Barbara L. Cool ◽  
Lemma Kifle ◽  
William Chiou ◽  
David A. Egan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Agarose Gel ◽  
Peptide Substrate ◽  
Screening Assay ◽  
Affinity Membrane ◽  
High Throughput Screening Assay ◽  
Compound Screening ◽  
Amp Activated Protein Kinase

A novel and innovative high-throughput screening assay was developed to identify both activators and inhibitors of AMP-activated protein kinase (AMPK) using microarrayed compound screening (μARCS) technology. Test compounds were arrayed at a density of 8640 on a polystyrene sheet, and the enzyme and peptide substrate were introduced into the assay by incorporating them into an agarose gel followed by placement of the gels onto the compound sheet. Adenosine triphosphate (ATP) was delivered via a membrane, and the phosphorylated biotinylated substrate was captured onto a streptavidin affinity membrane (SAM™). For detection, the SAM™ was removed, washed, and imaged on a phosphor screen overnight. A library of more than 700,000 compounds was screened using this format to identify novel activators and inhibitors of AMPK. ( Journal of Biomolecular Screening 2004:112-121)

scholarly journals A Scintillating Microplate Assay for the Assessment of Protein Kinase Activity

1998 ◽  
Vol 3 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Grace R. Nakayama ◽  
Michael P. Nova ◽  
Zahra Parandoosh
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinases ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Kinase Assay ◽  
Protein Kinase Activity ◽  
Discovery Research ◽  
Cell Extracts ◽  
Drug Discovery Research

Protein kinases, a class of enzymes that phosphorylate certain tyrosine, serine, and threonine residues, play an important role in cellular functions and are important targets in drug discovery research. Thus, it is of interest to develop a simple assay that can be used to measure protein kinase activity toward specific substrates and is suitable for the high throughput screening (HTS) of potential kinase inhibitors. The scintillation proximity concept has been successfully applied for measuring specific kinase activity using surfaces passively coated with a peptide substrate. In this study, we evaluated kinase assay performance on three ScintiStrip platforms: unmodified surface, streptavidin-coated surface, and streptavidin covalently attached to surface. The high affinity of streptavidin toward biotin-linked peptide substrates makes it a unique platform for measuring specific incorporation of radiolabeled phosphate into selected substrates of specific enzymes in the presence of others. Therefore, this assay may be used with cell extracts containing impure kinases as well as with purified enzymes. The scope of this assay was demonstrated with purified tyrosine kinases (e.g., p60c-src kinase) and A431 cell extracts. This scintillation proximity assay is universal, simple, rapid, accurate, and can be adapted for use with robotics for HTS.

A quantitative peptide array for evaluation of protein kinase activity

2008 ◽  
Vol 372 (1) ◽  
pp. 106-115 ◽  
Author(s):  
Xiaoming Han ◽  
Syuhei Shigaki ◽  
Takayuki Yamaji ◽  
Go Yamanouchi ◽  
Takeshi Mori ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Peptide Array

Effect of Thrombin on Phosphorylation of Platelet Membrane Proteins

1976 ◽  
Vol 35 (03) ◽  
pp. 635-642 ◽  
Author(s):  
M Steiner
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Qualitative Change ◽  
Protein Kinase Activity ◽  
Protein Substrates ◽  
Phosphoprotein Phosphatase ◽  
Progressive Loss ◽  
Platelet Membranes ◽  
Endogenous Protein ◽  
Considerable Loss

SummaryThe effect of thrombin on the phosphorylating activity of platelet membranes was compared to that of trypsin. Preincubation of non-32P phosphorylated platelet membranes with or without either of these two enzymes resulted in a considerable loss of membrane protein kinase activity which was most severe when trypsin was used. Protein kinase activity and endogenous protein acceptors decreased in parallel. 32P-phosphorylated membranes showed a slow but progressive loss of label which was accelerated by trypsin. Thrombin under these conditions prevented the loss of 32P-phosphate. These results are interpreted to indicate a thrombin-induced destruction of a phosphoprotein phosphatase. The protein kinase activity of phosphorylated platelet membranes using endogenous or exogenous protein substrates showed a significant reduction compared to non-phosphorylated membranes suggesting a deactivation of protein kinase by phosphorylation of platelet membranes. Neither thrombin nor trypsin caused a qualitative change in the membrane polypeptides accepting 32P-phosphate but resulted in quantitative alterations of their ability to become phosphorylated.

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